3 research outputs found
Phase-Retrieved Tomography enables imaging of a Tumor Spheroid in Mesoscopy Regime
Optical tomographic imaging of biological specimen bases its reliability on
the combination of both accurate experimental measures and advanced
computational techniques. In general, due to high scattering and absorption in
most of the tissues, multi view geometries are required to reduce diffuse halo
and blurring in the reconstructions. Scanning processes are used to acquire the
data but they inevitably introduces perturbation, negating the assumption of
aligned measures. Here we propose an innovative, registration free, imaging
protocol implemented to image a human tumor spheroid at mesoscopic regime. The
technique relies on the calculation of autocorrelation sinogram and object
autocorrelation, finalizing the tomographic reconstruction via a three
dimensional Gerchberg Saxton algorithm that retrieves the missing phase
information. Our method is conceptually simple and focuses on single image
acquisition, regardless of the specimen position in the camera plane. We
demonstrate increased deep resolution abilities, not achievable with the
current approaches, rendering the data alignment process obsolete.Comment: 21 pages, 5 figure
Demonstrating Improved Multiple Transport-Mean-Free-Path Imaging Capabilities of Light Sheet Microscopy in the Quantification of Fluorescence Dynamics
Optical microscopy constitutes, one of the most fundamental paradigms for the understanding of complex biological mechanisms in the whole-organism and live-tissue context. Novel imaging techniques such as light sheet fluorescence microscopy (LSFM) and optical projection tomography (OPT) combined with phase-retrieval algorithms (PRT) can produce highly resolved 3D images in multiple transport-mean-free-path scales. Our study aims to exemplify the microscopic capabilities of LSFM when imaging protein dynamics in Caenorhabditis elegans and the distribution of necrotic cells in cancer cell spheroids. To this end, we apply LSFM to quantify the spatio-temporal localization of the GFP-tagged aging and stress response factor DAF-16/FOXO in transgenic C. elegans. Our analysis reveals a linear nuclear localization of DAF-16::GFP across tissues in response to heat stress, using a system that outperforms confocal scanning fluorescent microscopy in imaging speed, 3D resolution and reduced photo-toxicity. Furthermore, we present how PRT can improve the depth-to-resolution-ratio when applied to image the far-red fluorescent dye DRAQ7 which stains dead cells in a T47D cancer cell spheroid recorded with a customized OPT/LSFM system. Our studies demonstrate that LSFM combined with our novel approaches enables higher resolution and more accurate 3D quantification than previously applied technologies, proving its advance as new gold standard for fluorescence microscopy
Controlling cell adhesion via replication of laser micro/nano-textured surfaces on polymers
The aim of this study is to investigate cell adhesion and viability on highly rough polymeric surfaces with gradient roughness ratios and wettabilities prepared by microreplication of laser micro/nano-textured Si surfaces. Negative replicas on polydimethylsiloxane as well as positive ones on a photocurable (organically modified ceramic) and a biodegradable (poly(lactide-co-glycolide)) polymer have been successfully reproduced. The final culture substrates comprised from forests of micron-sized conical spikes exhibiting a range of roughness ratios and wettabilities, was achieved by changing the laser fluence used to fabricate the original template surfaces. Cell culture experiments were performed with the fibroblast NIH/3T3 and PC12 neuronal cell lines in order to investigate how these surfaces are capable of modulating different types of cellular responses including, viability, adhesion and morphology. The results showed a preferential adhesion of both cell types on the microstructured surfaces compared to the unstructured ones. In particular, the fibroblast NIH/3T3 cells show optimal adhesion for small roughness ratios, independent of the surface wettability and polymer type, indicating a non-monotonic dependence of cell adhesion on surface energy. In contrast, the PC12 cells were observed to adhere well to the patterned surfaces independent of the roughness ratio and wettability. These experimental findings are correlated with micromechanical measurements performed on the unstructured and replicated surfaces and discussed on the basis of previous observations describing the relation of cell response to surface energy and rigidity. © 2011 IOP Publishing Ltd